We have evaluated different approaches to modulate the drug release profile from nanoparticles. First we synthesized triblock copolymers PCL-PEG-PCL with different molecular weights of PEG. Literature suggests that PCL block length affect the release profile of hydrophobic molecules since hydrophobic-hydrophobic interaction between the drug and polymer chains. Therefore, we evaluated the effect of hydrophilic chain length on the release profile of hydrophobic molecule. Small molecules can easily diffuse out of the polymeric chain as the drug release mechanism from the polymeric matrix. We found that there was no significant effect on the release pattern of triamcinolone acetonide from triblock copolymer nanaoparticles with different molecular weight of PEG. However, yet slower release observed from PEG of mw=2000 in comparison to PEG of mw=8000. Therefore, we seleacted PEG=2000 molecular weight for the synthesis of pentablock polymers.
We synthesized pentablock copolymers containing PEG, PCL and PLA blocks. Each block has its unique properties and the well known fact is that block copolymers can retain the properties of individual blocks. PEG is well known for its non-antigenic and non immunogenic nature. In addition, because of its hydrophilic nature it facilitates the diffusion of water into nanoparticles matrix and provides diffusion mediated drug release from nanoparticles. PCL is hydrophobic biodegradable polyester which enables high permeability for small molecules. Being hydrophobic in nature it also provides good encapsulation efficiency to lipophilic drugs via hydrophobic interactions. However, it exhibits very high crystallinity that is responsible for high burst release. PLA can influence the crystallinity of PCL. We wanted to have all these properties in a single polymer to prepare an ideal drug delivery system for delivery of steroids. Therefore, we optimize block ratio of PCL/PLA by synthesizing series of pentablock polymers. Only the discussed blocks have shown the significant difference in drug release profile. Our research group tries to improve the limitations of triblock polymers i.e. PCL-PEG-PCL by incorporating the additional block of PLA at the end by covalent conjugation, i.e. PLA-PCL-PEG-PCL-PLA. As we discussed in the manuscript existing triblock copolymers have limitation of burst release of drug from the nanoparticles. Burst release mainly responsible for the high crystallinity of PCL and surface adsorbed drug. For the first time we described the strategy how covalently conjugated PLA block can reduce the crystallinity of PCL-PEG-PCL and affect the drug release profile of triamcinolone acetonide. For the reference, we synthesized the triblock copolymers as well with same molecular weight of PCL. We characterize the triblock copolymers for crystallinity and drug release profile. We evaluated and compared the effect on crystallinity and drug release profile of pentablock polymers having the same molecular weight of PCL. We found that we are successful in changing the crystalline properties of triblock polymers by synthesizing pentablock polymers, which has altered the release profile of triamcinolone acetonide as well. Moreover, pentablock copolymer based nanoparticles exhibited minimal burst release profile. Therefore, we claimed that we synthesized novel pentablock polymers PLA-PCL-PEG-PCL-PLA, which have better properties compared to respective triblock polymers and can, provide sustained release profile for longer duration without producing significant burst release.
Further we evaluated the pentablock copolymers for long term delivery of timolol. Our earlier attempt of modulating the sustained release profile of timolol from PEG-PCL-PEG hydrogel by adding the polymeric additives did not show significant improvement (Mishra et al., 2011c). Therefore, we synthesized pentablock copolymer based thermosensitive hydrogel i.e. PEG-PCL-PLA-PCL-PLA. We found that pentablock copolymer based hydrogel have better reproducibility in terms od sol-gel transitive reversion compared to triblock polymer based hydrogels. However, to further achieve long term release profile we evaluated the composite approach. We suspended the nanoparicles formulation in to thermosensitive hydrogel solution. We found that drug release profile of timolol from both nanoparticles and thermosensitive gel alone was faster compared to the combination of both formulations. Since, timolol is a hydrophilic molecule diffusion of molecules from the gel was faster. In addition, nanoparticles suffer from the surface adsorb drug that is responsible for initial burst release. Our combination approach has significantly reduces the drug release rate because of the fact that drug has to travel longer through the polymeric matrix in the case of composite formulation.
Further, we evaluated the degradation kinetics of different pentablock copolymers. We performed the hydrolytic and enzymatic degradation of pentablock polymer based nanoparticles and thermosensitive hydrogel. We found that hydrolytic degradation of formulation was much slower compared to enzymatic degradation. These results could be explained by the fact that since PCL is very hydrophobic molecule the hydrolytic degradation is very slow. However, lipase enzyme can specifically degrades the PCL chains and low molecular weight PLLA chains. Therefore, we found faster degradation of PLLLA containing pentablock polymers compared to PDLLA containing structure.
Finally, we evaluated the potential of thermosensitive hydrogel copolymers in the long term delivery of sensitive protein molecules. We also evaluated the effect of size of the macromolecules in the release profile by utilizing FITC-dextran of molecular weights. We found that size of the molecules have significant effect on the release profile with smaller size molecules diffuses faster compared to larger size. These results are in agreement with our previous results with small molecules. We found that small molecules such as steroids having different hydrophobicity and log P values did not show significant difference in the release pattern because all are having the similar size. Since, thermosensitive hydrogels and nanoparticles are porous structures size of the molecules have highest impact on the release rate of molecules compared to other parameters. Long chains of large molecules get trapped in the polymer matrix that provides hindrance to the diffusion of molecules whereas small molecules can easily diffuse out of the pores of the polymer matrix. Moreover, we evaluated the biological activity and structural stability of sensitive molecules released from the thermosensitive hydrogel formulation. Since, hydrogel preparation does not involve any harsh treatment such as use of organic solvent or sonication, protein molecules can retain the structural integrity and activity while preparation of formulation. In addition, hydrogel can retard the direct contact of protein molecules with the water molecules that further minimizes the degradation of protein molecules inside the formulation. We also found that structural integrity and biological activity of lysozyme was maintained in the release medium during the entire period. Therefore, we conclude that our novel pentablock copolymers based thermosensitive hydrogel and nanoparticles have potential for long term delivery of small as well as macromolecules for the treatment of chronic ocular diseases where sustained levels of therapeutic molecules are required.
Recommendations
Pentablock copolymers can be further evaluated for the delivery of sensitive molecules such as proteins and antibodies. Major challenge for controlled delivery of therapeutic macromolecules is to maintain structural integrity and biological activity of protein for prolonged periods. These copolymers are amphiphilic in nature which can assist in optimizing the release profile of therapeutic macromolecules by varying the molecular weight of hydrophilic and hydrophobic blocks. Our dual approach of nanoparticles suspended in a thermosensitive gel can eliminate the burst release of drugs due to longer diffusion pathway of drug molecules through both nanoparticles and gel layers. Such innovative system can be introduced in to episcleral space through subconjunctival administration or in the intra vitreal cavity. Such a delivery system may result in prolonged duration of action, thereby completely eliminates the need for repeated intravitreal injections. Moreover, uptake of nanoparticles could be enhanced by functionalizing the surface groups such that the surface modified nanoparticles might be recognized by peptide transporter present on the basolateral side of the RPE and Bruch’s membrane. Intravitreal administration of surface modified nanoparticles suspended in thermosensitive gel may provide prolonged delivery with improved conformational stability and targeted delivery of therapeutic macromolecules. Scientific and clinical impact of developing a controlled polymeric delivery of therapeutic macromolecules for age related macular degeneration treatment may cause a paradigm shift in the treatment of retinal diseases. Our composite formulations may serve as a platform for delivery of other therapeutic proteins, peptides, siRNA, antibody and fragments. Subconjunctival injection of optimized formulation will overcome common side effects associated with intravitreal injections. Investigation would also provide insight into intravitreal pharmacokinetics of macromolecules upon administration of controlled release formulation, which would provide new mechanistic approaches to biomaterial design.
